A large number of human and experimental cancers display genetic alterations that activate G1-control kinases (CDK4 and CDK6). In this process, aberrant levels"ofD-type cyclins provide a growth advantage over normal cells. Whereas a role for cyclin D1 and D2 in cell proliferation have been established, recent data suggests that cyclin D3 (eye D3) plays additional roles in differentiation and growth arrest. This data correlates well with our preliminary results suggesting a role of eye D3 as a negative regulator of keratinocyte proliferation. Notably, overexpression of this cyclin results in inhibition of tumor development and decreased malignant progression to squamous cell carcinomas (SCC). Analysis of primary keratinocytes shows that overexpression of eye D3 results in strong reduction of the eye D2 protein levels, whereas elevated levels of eye D2 was observed in eye D3 null mice. Thus, we have hypothesized that eye D3 negatively regulate keratinocyte proliferation through a posttranslational mechanism downregulating eye D2. Supporting this hypothesis, cell lines derived from keratinocytes, papillomas and SCC, showed increased eye D3 stability in all but the SCC cell lines, whereas cell lines derived from SCC showed elevated stability of eye D1 suggesting that these two cyclins play opposing roles. To determine the potential application of D-type cyclins as target for therapeutic intervention it is essential to understand the role of each member. The work performed for this application has led to a number of relevant questions related to the roles of D-type cyclins in neoplastic development and epidermal homeostasis. However, in order to remain focused, we will concentrate on the role of eye D3 and eye D2 in tumorigenesis. Based on the preliminary results obtained for this application, we proposed two hypotheses: 1- Cyc D3 expression inhibits tumorigenesis through a posttranslational mechanism that results in decreased levels of eye D2 changing the proliferative capacity of epidermal cells. 2- Cyc D3 expression inhibits carcinogenesis by positive regulation of the differentiation process. In order to investigate these hypotheses, we propose the following specific aims: SA 1: To determine the effect of unbalanced expression of D-type cyclins in tumorigenesis. SA 2: To determine the posttranslational mechanism that regulates the levels of cyclin D2. SA 3: To determine the role of cyclin D3/CDK6 complexes in normal and neoplastic proliferation, and keratinocyte differentiation.